SUMOs (small ubiquitin-like modifiers) are ubiquitin-like proteins (Ubls) that become conjugated to substrates through a pathway that is biochemically similar to ubiquitination. SUMOylation is involved in many cellular processes, including DNA metabolism, gene expression and cell cycle progression. Clinical studies suggest that SUMOylation plays important roles in disease processes, including diabetes, viral infection and carcinogenesis. Notably, there are a broad variety of SUMOylated target proteins, many of whose modification may be mis-regulated in human diseases. While SUMOylation is thus broadly implicated in the pathology of these diseases, one major challenge in this field is to understand its role at the level of individual substrates and of the cellular processes that they regulate. We are particularly interested in the capacity of SUMOylation to control cell division and nuclear transport. Vertebrate cells express three major SUMO paralogues (SUMO-1-3): Mature SUMO-2 and -3 are 95% identical to each other, while SUMO-1 is 45% identical to SUMO-2 or -3. (Where they are functionally indistinguishable, SUMO-2 and -3 will be collectively called SUMO-2/3.) Like ubiquitin, SUMO-2/3 can be assembled into polymeric chains through the sequential conjugation of SUMOs to each other. SUMO-1 appears less likely to form similar chains, and and might only become conjugated to the end of SUMO-2/3 chains as a chain terminator. A large number of SUMOylation substrates have been identified. SUMOylation promotes a variety of fates for individual targets, dependent upon protein itself, the conjugated paralogue and whether the conjugated species contains a single SUMO or SUMO chains. SUMOylation is dynamic due to rapid turnover of conjugated species by SUMO proteases. Both processing and deSUMOylation are mediated by the same family of proteases (called Ubl specific proteases (Ulp) in yeast and Sentrin-specific proteases (SENP) in vertebrates), which play a pivotal role in determining the spectrum of SUMOylated species. There are two yeast Ulps (Ulp1p and Ulp2p/Smt4p), and six mammalian SENPs (SENP1, 2, 3, 5, 6, and 7). SENP1, 2, 3 and 5 form a Ulp1p-related sub-family, while SENP6 and 7 are more closely related to Ulp2p. Yeast Ulps have important roles in mitotic progression and chromosome segregation. We have defined the enzymatic specificity of the vertebrate SENP proteins and analyzed their key biological roles, particularly the functions of SENP3 and SENP5 in ribosome biogenesis and the function of SENP6 in kinetochore assembly. Ulp1p localizes to nuclear pore complexes (NPCs) and is encoded by an essential gene; it is important for SUMO processing, nucleocytoplasmic trafficking and late steps in the ribosome biogenesis pathway. Ribosome biogenesis occurs largely within the nucleolus. It is a major metabolic expense and a critical point of cellular regulation during both cell growth and cancer progression. Human SENP3 and SENP5 are nucleolar Ulp1p-like SUMO proteases that are closely related to each other, and that have enzymatic specificity for SUMO-2/3 over SUMO-1. B23/nucleophosmin is an abundant 37-kD phosphoprotein that shuttles between the nucleolus and cytoplasm, which is implicated in many cellular processes, including ribosome biogenesis and control of the Arf-MDM2-p53 pathway. B23/nucleophosmin is often overexpressed in solid tumors and has been strongly linked to hematopoietic malignancies. We found that B23/nucleophosmin is essential for the stable accumulation of SENP3 and SENP5. Importantly, depletion of SENP3 and SENP5 causes defects in ribosome biogenesis closely reminiscent of those observed in the absence of B23/nucleophosmin, suggesting that control of SUMO deconjugation through SENP3 and SENP5 may be a major facet of B23/nucleophosmin function. To understand the role of SENP3 at a biochemical level, we have characterized its protein interaction partners within Xenopus egg extracts (XEEs). We found that SENP3 binds stably to three proteins that show sequence similarity to yeast ribosome assembly factors Rix1p, Ipi1p and Ipi3p. Mammalian homologues of these SENP3-interacting proteins are essential for ribosome biogenesis in tissue culture cells, further suggesting that they are previously unrecognized vertebrate homologues of the yeast Rix1 complex. Ran is a small GTPase that binds to a family of nuclear transport receptors in its GTP-bound state. Ran-GTP binding regulates the cargo binding and release of these receptors, so that the differential levels of Ran-GTP in the cytoplasm and nucleus determine the directionality of transport. SENP3 and the Rix1 complex bound 60S ribosomal particles through B23/Nucleophosmin under high Ran-GTP conditions. Under low Ran-GTP conditions, B23/nucleophosmin was lost and they bound instead to RanBP5, a nuclear import receptor. While there is no evidence that yeast Rix1 activity is integrated with Ulp1p function, our findings demonstrate that SENP3 and the vertebrate Rix1 complex are coupled, both physically and functionally. Our data also suggest that B23/nucleophosmin and the Ran pathway regulate SENP3 and the Rix1 complex in a novel, antagonistic fashion. While humans possess two NPC-associated SENPs, SENP1 and SENP2, we found that frogs possess a single member of this family, xSENP1. We are currently exploiting this difference to analyze the function of NPC-bound SUMO proteases. We have determined the interaction partners of this enzyme throughout the cell cycle using Xenopus egg extracts (XEEs), and are using XEEs to analyze both the function of xSENP1 and the role of its interacting partners. Yeast Ulp2p is nucleoplasmic and not essential for vegetative growth, but it is important for chromosome segregation. Ulp2p acts particularly in disassembly of poly-SUMO chains. We have demonstrated that human SENP6 is a vertebrate Ulp2p-related enzyme that similarly prefers substrates containing multiple SUMO-2/3 moieties. PolySUMOylated species are also substrates of SUMO-targeted ubiquitin ligases (STUbLs), leading to their proteasomal degradation. The RNF4 protein is a major STUbL in mammalian cells. We analyzed the mitotic role of SENP6 in mammalian cells, and found that it is essential for accurate chromosome segregation. Faulty chromosome alignment reflected the loss of inner kinetochore proteins, including components of the CENP-H/I/K and CENP-O complexes. Importantly, we found that the CENP-H and -I proteins were quantitatively degraded in the absence of SENP6 through a mechanism that requires both RNF4 and proteasome-mediated proteolysis. Together, these findings demonstrate a novel function of the SUMO pathway in inner kinetochore assembly, which finely balances the incorporation and degradation of components of the inner plate. We are currently analyzing other aspects of SENP6 mitotic function, including its role in chromosome morphology. At the same time, we are interested in defining other events regulated by SENP6 and RNF4. Toward this end, we are using XEEs to identify both polySUMOylated proteins and the cellular proteins that bind to SUMO chains.